The ubiquitously expressed human CDK8 kinase is a potent oncogene, and is required for mammalian development. CDK8 kinase activity is essential for its biological function; however, few kinase substrates have been identified. A basic first ste in understanding the biological role for any kinase is to identify the substrates that it modifies (and therefore regulates). The experiments described herein will address these important issues using a powerful combination of approaches; this project involves collaboration between two labs whose diverse areas of expertise will synergize to tackle this important yet challenging problem. To identify CDK8 kinase targets, we will perform comparative SILAC-based phosphoproteomic analyses in colon cancer cell lines treated (or not treated) with a potent and highly selective CDK8 kinase inhibitor. We will also complete comparative phosphoproteomic analyses in cells that lack CDK8, or express wild-type CDK8, kinase-dead CDK8, or constitutively active CDK8. Only a handful of CDK8 kinase substrates have been identified (e.g. histone H3, E2F1), yet each is known to play major roles in controlling transcription in both general and gene-specific ways. Because CDK8 reversibly associates with the Mediator complex (a genome-wide regulator of RNA polymerase II), it is recruited to regulatory loci throughout the genome. A global, unbiased assessment of CDK8 kinase targets will provide unprecedented insight into how the CDK8 kinase regulates transcription. Moreover, the identified CDK8 substrates may reveal new targets and strategies to block CDK8-dependent pathways in cancer cells.